T4溶菌酶在毕赤酵母中的诱导表达及抑菌活性测定

T4溶菌酶在毕赤酵母中的诱导表达及抑菌活性测定

郝雯静, 李刚强, 徐妙云, 魏昭荣, 陈海敏, 刘德虎^*, 艾铁民^*^*

1.北京大学药学院, 北京 100083;
2.中国农业科学院生物技术研究所, 北京 100081

收稿日期:2006-11-14 修回日期:2007-02-10 出版日期:2007-03-15 发布日期:2007-03-15
通讯作者: 刘德虎*, 艾铁民**

Induction and expression of T4 lysozyme gene in Pichia pastoris

Wen-Jing Hao, Gang-Qiang Li, Miao-Yun Xu, Zhao-Rong Wei, Hai-Min Chen, De-Hu Liu^*, Tie-Min Ai^*^*

1. School of Pharmaceutical Sciences, Peking University, Beijing 100083, China;
2. Institute of Biotechnology Research, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Received:2006-11-14 Revised:2007-02-10 Online:2007-03-15 Published:2007-03-15
Contact: De-Hu Liu*, Tie-Min Ai**

摘要/Abstract

摘要：

目的利用毕赤酵母诱导表达T4溶菌酶蛋白并测定其抗菌活性。方法 T4溶菌酶(T4 lysozyme)基因以N端融合的方式被准确插入到质粒pPIC9K的EcoR I-Not I位点内,得到分泌型重组表达载体pPIC9K-T4L。该载体首先经限制性内切酶Sal I酶切,进而采用电击方法将线性化的重组质粒DNA导入到毕赤酵母中。经过梯度筛选得到多个单拷贝和多拷贝转化重组子。随机挑取部分重组子PCR扩增阳性克隆,经菌体培养和甲醇诱导后获得了分泌表达,表达产物存在于培养上清液中。结果表达蛋白经琼脂孔扩散抗菌实验显示抑菌圈明显; 重组蛋白对金黄色葡萄球菌与肺炎链球菌均具有显著抑制作用; 多拷贝与单拷贝重组表达子没有抗菌活性差异与蛋白表达量的差异; 加热煮沸对于T4溶菌酶蛋白的抗菌活性无明显影响。结论 T4溶菌酶在毕赤酵母中得以成功诱导与表达; 表达产物不受拷贝数影响并具热稳定性。

关键词: T4溶菌酶, T4溶菌酶, T4溶菌酶, 毕赤酵母, 毕赤酵母, 毕赤酵母, 电击转化, 电击转化, 电击转化, 表达, 表达, 表达, 抗菌活性, 抗菌活性, 抗菌活性

Abstract:

Aim To induce and express the T4 lysozyme in Pichia pastoris and test the antibacterial activity of the protein. Methods T4 lysozyme gene was inserted into expression vector pPIC9K of Pichia pastoris with the fusion at N terminal. The recombinant plasmid was digested by Sal I and then introduced into prepared GS115 competent cells by electroporation. Positive clone and multiple inserts were screened. The secreted proteins in the supernatants were tested. In the agar holes diffusion assay, our expressed protein showed significant antibacterial circles. Results T4 lysozyme protein inhibited the growth of staphylococcus aureus and streptococcus Pneumoniae. There was no difference in the bactericidal activity and the amount of protein expression between the single and multiple copies. The antibacterial activity of expressed protein remained the same during the heat stability test. Conclusion T4 lysozyme was successfully induced and expressed in Pichia pastoris. There is no relationship between copy number and expression. T4 lysozyme protein is heat stable.

Key words: T4 lysozyme, T4 lysozyme, Pichia pastoris, Pichia pastoris, Electroporation, Electroporation, Expression, Expression, Antibacterial activity, Antibacterial activity

中图分类号:

R927.3

Supporting:

^*Corresponding author. Tel.: 86-10-68919865, ^**Tel.: 86-10-82802685

郝雯静, 李刚强, 徐妙云, 魏昭荣, 陈海敏, 刘德虎^*, 艾铁民^*^*. T4溶菌酶在毕赤酵母中的诱导表达及抑菌活性测定[J]. .

Wen-Jing Hao, Gang-Qiang Li, Miao-Yun Xu, Zhao-Rong Wei, Hai-Min Chen, De-Hu Liu^*, Tie-Min Ai^*^*. Induction and expression of T4 lysozyme gene in Pichia pastoris[J]. .

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